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Mfn1 Structures Reveal Nucleotide-Triggered Dimerization Critical for Mitochondrial Fusion

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Mfn1 Structures Reveal Nucleotide-Triggered Dimerization Critical for Mitochondrial Fusion HHS Public Access Author manuscript Author ManuscriptAuthor Manuscript Author Nature. Manuscript Author Author manuscript; Manuscript Author available in PMC 2017 July 23. Published in final edited form as: Nature. 2017 February 16; 542(7641): 372–376. doi:10.1038/nature21077. Mfn1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion Yu-Lu Cao1, Shuxia Meng2, Yang Chen1, Jian-Xiong Feng1, Dong-Dong Gu1, Bing Yu1, Yu- Jie Li1, Jin-Yu Yang1, Shuang Liao1, David C. Chan2, and Song Gao1 1State Key Laboratory of Oncology in South China, Collaborative Innovation Center for Cancer Medicine, Sun Yat-sen University Cancer Center, Guangzhou, 510060, China 2Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125 Abstract Mitochondria are double-membrane organelles with varying shapes influenced by metabolic conditions, developmental stage, and environmental stimuli1–4. Their dynamic morphology is realized through regulated and balanced fusion and fission processes5, 6. Fusion is crucial for the health and physiological functions of mitochondria, including complementation of damaged mitochondrial DNAs and maintenance of membrane potential6–8. Mitofusins (Mfns) are dynamin- related GTPases essential for mitochondrial fusion9, 10. They are embedded in the mitochondrial outer membrane and thought to fuse adjacent mitochondria via concerted oligomerization and GTP hydrolysis11–13. However, the molecular mechanisms behind this process remains elusive. Here we present crystal structures of engineered human Mfn1 containing the GTPase domain and a helical domain in different stages of GTP hydrolysis. The helical domain is composed of elements from widely dispersed sequence regions of Mfn1 and resembles the Neck of the bacterial dynamin-like protein. The structures reveal unique features of its catalytic machinery and explain how GTP binding induces conformational changes to promote G domain dimerization in the transition state. Disruption of G domain dimerization abolishes the fusogenic activity of Mfn1. Moreover, a conserved aspartate trigger was found in Mfn1 to affect mitochondrial elongation, likely through a GTP-loading-dependent domain rearrangement. Based on these results, we propose a mechanistic model for Mfn1-mediated mitochondrial tethering. Our study provides important insights in the molecular basis of mitochondrial fusion and mitofusin-related human neuromuscular disorders14. Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:http://www.nature.com/authors/editorial_policies/license.html#terms Contact information: Correspondence: [email protected]. Author Contributions S.G. and D.C.C. conceived the project. Y.-L.C. made the constructs, purified proteins, and performed crystallographic and biochemical experiments. S.M. carried out mitochondrial elongation assays. Y.C. did ITC measurements and helped with collection of X-ray diffraction data. J.-X.F., B.Y and Y.-J.L. did cloning and purification for some of the Mfn1IM mutants. D.-D.G. performed some of the SEC-RALS experiments, D.-D.G., J.-Y.Y. and S.L. helped with crystallization experiments. Y.-L.C. S.L. and S.G. solved the structures. Y.-L.C., D.C.C. and S.G. wrote the paper. Author Information The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to S.G. ([email protected]). Cao et al. Page 2 We constructed an internally modified human Mfn1 (Mfn1IM) composed of the GTPase (G) Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author domain (residues 75–336) and a four-helix-bundle that we term helical domain 1 (HD1, Fig. 1a, 1b, Extended Data Fig. 1a–g and Extended Data Table 1). The G domain contains a central eight-strand β-sheet surrounded by eight α-helices. Compared to the canonical GTPase Ras, the G domain of Mfn1 has two extra lobes that shield the nucleotide binding pocket, and a specific short α-helix (α2’G) sitting between α4G and β6G (Fig. 1c). Lobe 1, containing two β-strands (β1’G and β2’G) and an α-helix (α1’G), is located between β2G and β3G, whereas Lobe 2, consisting of an α-helix (α3’G) and loop, is located between β6G and α5G (Extended Data Fig. 2a). The four α-helices of the HD1, derived from widely dispersed sequence regions, form a vast and conserved hydrophobic network (Fig. 1a, d and Extended Data Fig. 2b). HD1 is connected to the G domain via R74 at the C-terminal end of α2H and K336 between α5G and α3H (Fig. 1b). The N terminus of α1H substantially contacts the G domain (Extended Data Fig. 2c–e). On the other side of HD1, part of the artificial linker folds into an α-helix extending α3H (Fig. 1b). This is in agreement with the secondary structure prediction for the replaced residues (Extended Data Fig. 3), suggesting that α3H may be longer in full-length Mfn1. 15–20 The overall topology of Mfn1IM is typical of the dynamin superfamily (Extended Data Fig. 4). Apart from the G domain, the Mfn1IM HD1 is particularly consistent with the Neck of the bacterial dynamin-like protein from Nostoc punctiforme (BDLP), which was suggested to mediate membrane fusion in bacteria21 (Extended Data Fig. 2a, 4, 5a). Given the compact organization of HD1 and the predicted secondary structure (Extended Data Fig. 3), the missing portion of Mfn1 (excluding TM) from Mfn1IM is likely to fold into a helix- rich domain resembling the Trunk and Paddle of BDLP21. We term this putative region helical domain 2 (HD2). When bound to tubulated liposomes in the presence of GMPPNP, BDLP bears G domain- Neck and Neck-Trunk rearrangements via so-called Hinge 2 and Hinge 122. Intriguingly, K336 of Mfn1IM exactly overlaps with BDLP R327 at Hinge 2b, whilst Mfn1IM G309 and R74 also have counterparts in BDLP (G309 in Hinge 2b and G68 in Hinge 2a) at equivalent positions (Fig. 1e). Mutation of these Hinge 2-related residues diminished GTPase activity and mitochondrial elongation, although the G domain and HD1 exhibited only limited relative movement in different nucleotide-loading states (Extended Data Fig. 5b–e). Like in dynamins, the potential Hinge 1 between HD1 and predicted HD2 (YSVEER368–373 and EEEIAR692–697) lacks overall conservation among mitofusins (Extended Data Fig. 3). A proline insertion in EEEIAR692–697 abolished mitochondrial elongation activity (Extended Data Fig. 5e, f). Altogether, full-length Mfn1 possibly undergoes conformational changes similar to BDLP when mediating mitochondrial outer membrane (OMM) fusion via aforementioned hinges. G1-G4 elements of GTPases are essential for binding and hydrolysis of GTP (Fig. 2a). Strikingly, in the nucleotide-free (apo) Mfn1IM structure, the nucleotide-binding pocket is occupied by the bulky side chain of W239 from G4, a residue conserved only in mitofusins and BDLP (Fig. 2b). Loading of GTP drives W239 away, causing it to wedge into a wide hydrophobic groove formed by M249 from α4G and F282 from β6G. This rearrangement allows the suitable positioning of N237 and D240 to dock the guanine base (Fig. 2b). Nature. Author manuscript; available in PMC 2017 July 23. Cao et al. Page 3 Mutation of W239 to alanine abolished nucleotide-binding and GTPase activity of Mfn1IM Author ManuscriptAuthor Manuscript Author Manuscript Author Manuscript Author (Fig. 2c, 2d). Both Mfn1W239A and the corresponding Mfn2 mutant Mfn2W260A were nonfunctional for mitochondrial elongation (Fig. 2e), manifesting the importance of this G tryptophan switch. When accommodating a nucleotide, Mfn1IM utilizes α2’ to loosely buttress the guanine from a vertical orientation, and a large area of the nucleotide is thus exposed. This feature is shared by BDLP but not with Dynamin-121, 23, where the nucleotide is tightly wrapped (Extended Data Fig. 5g). Whereas the apo and GTP structures are monomeric, Mfn1IM forms a homodimer in the − presence of the transition state mimic GDP•AlF4 (Extended Data Fig. 6a). Dimerization is mediated by association of the G domains across the nucleotide binding pocket, and the HD1s protrude in opposite directions from the dimer axis (Fig. 3a). Major interactions of this 995 Å2 ‘G interface’ include a pair of symmetrical, parallel aligned salt bridges between R238 in the G4 element and E209 in the loop between β3G and α3G (Fig. 3b). Flanking this central salt bridge pair, close in trans contacts are also observed between K99-E245, H144- E247, and H147-D251. In addition, the side chain of Y248 inserts into the groove between the Switch I and α1’G of the other molecule (Fig. 3b). G domain dimerization has been found in several dynamin superfamily members in the transition state of GTP hydrolysis23–25. Compared to the ∼2,500 Å2 G interface of Dynamin-123 involving extensive hydrogen bonds and hydrophobic associations, the substantially smaller G interface of Mfn1IM is dictated by charged interactions, and no in trans stabilization of the nucleotides is observed (Fig. 3c). To verify the functional relevance of G domain-mediated dimerization, we performed − mutagenesis studies on residues E209 and R238. Whereas GDP•AlF4 induced the formation of Mfn1IM dimers in analytical gel filtration coupled to right angle light scattering E209A R238A (RALS) assays, Mfn1IM and Mfn1IM failed to dimerize (Fig. 3d). Moreover, the two mutants lacked stimulated protein-concentration-dependent GTPase activity (Fig. 3e), even though they bound guanine nucleotides with wild-type affinity (Fig. 3f). These results suggest that GTPase activation is mediated by dimerization via the G interface. In addition, E209A R238A Mfn1IM and Mfn1IM showed suppressed liposome tethering activity in vitro (Extended Data Fig. 6b). Both Mfn1E209A and Mfn1R238A, as well as the corresponding Mfn2 mutants Mfn2E230A and Mfn2R259A, failed to elongate mitochondria (Fig. 3g). Thus, G domain association of mitofusins during the transition state of GTP hydrolysis is an indispensable step for OMM fusion. In addition, mutations of most other residues involved in the G interface also impinge dimerization, GTP hydrolysis and mitochondrial elongation to various extents (Extended Data Fig.
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